Cancer is a leading cause of morbidity in the world. Work has been carried out for many years on the identification of molecules that are characteristic of the disease. Many have been found to be unique in tumours or are overexpressed, altered or otherwise differing in cancer. Such tumour markers are used as targets in the many immunological approaches to cancer diagnosis and therapy.
The MUC1 protein is one such marker that is known to be overexpressed in malignant cells. This epithelial mucin is coded for by the MUC1 gene. It is not a classic extracellular complex mucin, such as those found as major components of the mucous layers covering the gastro-intestinal and respiratory tracts, but is a transmembrane molecule, expressed by most glandular epithelial cells. The protein consists of a number of distinct regions, including an N-terminus with a putative signal peptide and degenerate tandem repeats, a transmembrane region and a C-terminus cytoplasmic tail. The major portion of the protein is the tandem repeat region. This consists of degenerate repeats of the unique peptide sequence APDTRPAPGSTAPPAHGVTS. The number of repeats varies with the allele, thus making the gene and protein highly polymorphic. MUC1 is also referred to as MUC-1, mucin 1, muc-1, episialin, peanut-reactive urinary mucin (PUM), polymorphic epithelial mucin (PEM), CD227, epithelial membrane antigen (EMA), DF3 antigen and H23 antigen.
MUC1 mucin is restricted to the apical cell surface by interactions with the microfilament network. Although MUC1 is widely expressed by normal glandular epithelial cells, the expression is dramatically increased when the cells become malignant. This has been well documented for breast and ovarian cancer, as well as some lung, pancreatic and prostate cancers. Recently it has also been shown that MUC1 is a valuable marker for bladder cancer and has been used for its diagnosis in a number of studies. Antibody studies have also shown not only that MUC1 is overexpressed in carcinomas but also that the pattern of glycosylation is altered. Thus, in the breast cancer mucin, glycosylation changes result in certain epitopes in the core protein being exposed which are masked in the mucin produced by the lactating mammary gland. These characteristics of MUC1 have been explored over the years in a number of immunotherapeutic approaches, mainly involving radiolabelled antibodies against breast and bladder cancers. Other attempts on active specific immunotherapies based on MUC1 have also taken place in animal models to investigate the efficacy of immunogens based on MUC1.
These immunotherapeutic approaches had some encouraging results and have led to clinical trials both for the vaccine therapies and the antibody treatments. These strategies however are not without problems. The radiolabelled antibody technique is limited to modest (millicurie) radiation doses since the long circulation time of radiolabelled antibodies makes bone marrow toxicity a problem. Another problem is the time period required to produce specific monoclonal antibodies. Additionally, recent attempts to use peptides instead of antibodies have resulted in molecules with very low affinity for the MUC1 mucin.
International patent application WO2004/081574 describes the generation of aptamers to a synthetic MUC1 peptide. The publication describes the generation of aptamers targeted either to a 60-mer peptide consisting of three copies of the repeat sequence APDTRPAPGSTAPPAHGVTS (SEQ ID NO: 12), or to the nine amino acid peptide APDTRPAPG (SEQ ID NO: 13) contained within this sequence. While this approach may generate useful aptamers, it is possible that alternative approaches may be useful in generating aptamers against different epitopes of the MUC1 peptide. Further, the glycosylation pattern of MUC1 in vivo is known to be altered in carcinomas, such that aptamers to an unglycosylated synthetic peptide may not be as efficient at binding to the peptide in vivo as alternative aptamers raised against the tumour-glycosylated MUC1 protein.
The present invention seeks to overcome or alleviate at least some of these problems with the identification of aptamers based on a recombinant MUC1 protein. In preferred embodiments, this recombinant protein has been glycosylated in a way similar to that utilised in nature, offering a further level of improvement in approaching the in vivo conditions in which our aptamers would operate (for example as therapeutics or diagnostics in the body).
The advantage of this invention in relation to existing inventions based on antibodies recognising MUC1 is that the aptamers provide much more specific and stronger binding to the MUC1 target protein, thus becoming more sensitive in recognising the protein in solution, thus offering the potential of significant reduction in the recognition levels of the tumour marker in current diagnostic assay, resulting in more sensitive assay to offer better prognostic/early diagnostic value. Furthermore, when compared with antibodies as therapeutics, aptamers offer little or no immunogenicity, better tumour penetration, rapid clearance through the kidneys and the urine and natural degradation after a certain amount of time.
The invention can be applied commercially in the diagnosis, imaging and treatment of cancer. It consists of a molecule that recognises a tumour marker, a molecule that is characteristic for the tumour cell or in a different form in tumour cells than in normal cells. This tumour marker is expressed on the surface of cancer cells, but it is also shed in the bloodstream. As such, the molecule(s) that constitute the invention have the potential to be used in diagnostic assays/blood tests to provide a prognosis and/or early diagnosis of tumour or metastatic disease. Furthermore, the molecule has the ability to carry to the surface of the tumour cells modalities that can effect cell kill. Thus, coupling of the molecule to chemotherapy or radiotherapy agents can take them specifically to the tumour cell, thus reducing side effects associated with generic treatments. Finally, coupling of the agent to fluorescent, near infrared, MRI or radionuclide ligands can create a powerful imaging agent that would significantly increase imaging sensitivity and image definition.